Analysis of thyroid function and related factors in narcolepsy patients

The loss of hypocretin is thought to be the main pathophysiological mechanism of narcolepsy. There is strong evidence that hypocretin is related to the regulation of endocrine functions and depression. To explore thyroid hormone levels in narcolepsy patients was our aim. In addition, further is to analyze the relationship between thyroid hormone levels and sleep quality, anxiety, and depression in narcolepsy patients. There are 40 patients with narcolepsy and 40 healthy controls (HCs) were conducted. Blood samples were explored for thyroid function. Correlation analysis between thyroid hormones and clinical characteristics of narcolepsy was performed using Pearson or Spearman. Narcolepsy patients had significantly lower free thyroxine (FT4) levels in comparison to controls (p < 0.001). No subject was diagnosed with primary hypothyroidism. There were 4 (10%) subjects with subclinical hypothyroidism. The serum FT4 levels were positively correlated with HAMA14 score (r = − 0.343, p = 0.030) by Pearson correlation analysis. The serum TSH levels and HAMD24 score (r = − 0.807 p ˂0.001), and ESS score (r = − 0.317, p = 0.046) both showed a negative correction. Hypocretin deficiency may be associated with the regulation of thyroid hormones in narcolepsy patients. The serum thyroid hormones may affect the severity and neuropsychological functions of narcolepsy patients.

local association networks, no objective sleepiness on the MSLT (mean sleep latency > 8 min), no medication use, and no other significant psychiatric, neurologic, or medical disorders.
The study was approved by the Ethics Committee of The Second Hospital of Shandong University, and all participants signed written informed consent prior to participation.This study was conducted in accordance with the Declaration of Helsinki.

Exclusion criteria
Exclusion criteria were as follows: (1) medical history of central nervous system demyelinating disease, central nervous system infection, and intracranial tumors; (2) patients with a history of metabolic illness(e.g.autoimmune liver disease, viral hepatitis, diabetes, and hypertension) were excluded; (3) patients with a history of 131 I treatment for hyperthyroidism, neck radiotherapy, thyroidectomy were excluded; (4) Exclusion of participants with comorbid other forms of sleep disorders (e.g.OSA, RLS); (5) Comorbid psychiatric disorders were excluded 20 .

Collecting general information
To assess the subjects' EDS symptoms, the Epworth sleepiness scale (ESS) was used by professional physicians 21 .

Laboratory assessment
After an overnight fast of 8 h, venous blood samples were collected from all subjects and serum free thyroxine (FT 4 ), free triiodothyronine (FT3) and thyroid stimulating hormone (TSH) levels were assessed according to standard operating procedures, the Architect i2000SR automated immunoassay system was used to measure FT 4 , FT3 and TSH 20 .We used the 125 I radioimmunoassay kit (Phoenix Pharmaceuticals, Belmont, CA, USA) to muesure the cerebrospinal fluid (CSF) hypocretin-1 levels.

Clinical and neuropsychological assessment
To measure the sleep quality, the Pittsburgh sleep quality index (PSQI) was used 20,22 .To measue the anxiety, the 14-item Hamilton Anxiety Scale (HAMA 14 ) was used 20 .While, to measure the depression symptoms of narcoelpsy patients, the 24-item Hamilton depression rating scale (HAMD 24 ) were used 20,23 .

Statistical analysis
All analyses were performed using SPSS 24.0 computer software 20 .The mean ± standard deviation (SD) was uesed to reprent the continuous variables.The interquartile range (M, P 25 , P 75 ) was used to present non-normally distributed data, which was tested by the student's t test.Mann-Whitney U test was used to test the normality of the data.To compare the groups, we used the Chi-square test or Fisher's exact test.Categorical data were expressed in amount (%) 20 .Spearman or Pearson for correlation analysis 20 .A P value of less than 0.05 was considered statistically significant.

Ethics statement
The study was approved by the Ethics Committee of The Second Hospital of Shandong University (No.2021141), and all participants signed written informed consent prior to participation.This study was conducted in accordance with the Declaration of Helsinki..

Demographics and clinical characteristics
A total of 40 patients with NT1 were included in the study according to the inclusion and exclusion criteria (Fig. 1).Biochemical characteristics and demographic data characteristics of the participants are presented in Table 1.

Comparison of thyroid hormones of NT1 patients and healthy subjects
The levels of FT4 in serum (p < 0.001) were significantly lower in patients with NT1 compared to the HC group.However, no differences were found between TSH (p = 0.906) or FT3 (p = 0.274) levels (Table 2).

The number of subjects in NT1 patients categorized by thyroid hormones
Table 3 shows the number of subjects in the NT1 group classified by FT4, FT3 and TSH levels.4 (10%) patients had higher than normal TSH levels.0 (0%) and 4 (10%) subjects had lower than normal FT3 and FT4 levels.No patient was diagnosed with primary hypothyroidism, compared to 4 (10%) subjects who were diagnosed with subclinical hypothyroidism.No hyperthyroidism was found in our study.

Correlation analysis of thyroid hormones and clinical characteristics of NT1
A statistically significant negative correlation was found between serum TSH levels and HAMD 24 score (r = − 0.807, P-value ˂0.001), and ESS score (r = − 0.317, P-value = 0.046).The serum FT 4 levels was positively correlated with HAMA 14 score(r = − 0.343, P-value = 0.030) by Pearson correlation analysis (Table 4).   reported that the serum levels of TSH were lower in patients with NT1.Szakacs et al. 24 considered that the median TSH was decreased in NT1 patients with cataplexy.Our results also suggested that the FT 4 was significantly decreased in NT1 patients.However, Chabas et al. 25 found no different in serum TSH and FT 4 levels.In addition, there was no difference in FT 3 and TSH levels between NT1 patients and the control group.Moreover, in an autopsy study, the thyrotropin releasing hormone-expressing neurons were unaffected in paraventricular nucleus (PVN) of patients with NT1 26 .Thyroid hormone changes in patients with NT1 remain unknown due to the small number of studies and conflicting results.CG-3703, a thyroid-releasing hormone (TRH) analog can increase wakefulness, suppressed both REM sleep, slow-wave sleep, and significantly reduce cataplexy in canine NT1 27,28 .However, there were four (10.0%) subjects with subclinical hypothyroidism in our study.Hypocretin can regulate the HPT axis, which could provide insights into the understanding of the broad symptom spectrum of NT1.Martinez-Orozco et al. 29 found an increased prevalence of autoimmune thyroid disease in NT1 patients, which may provide a valuable starting point for basic and applied research into the disease.However, autoimmune thyroid disease was not seen in our present study.

Orexin system and HPT axis
Several animal experiments have already demonstrated that the hypocretin system can be able to modulate the HPT axis in rodents.Kaufman et al. 30 found that the levels of thyroid hormone were lower in rats with LHA lesions.Besides, Suzuki et al. 31 found that the administration of TRH in the LHA can significantly enhance the anorexia effect.In addition, studies have demonstrated that peripheral administration of hypocretin-1 can inhibit the TRH release from the hypothalamus in rats 32 .Different mechanisms for the causal relationship between thyroid function and NT1 were suggested.The PVN can receive hypocretin input signals 33 , which is also the central point of the regulation of the HPT axis 34 .Hypophysiotropic neurons of the PVN of the hypothalamus expressing TRH project to the portal system, via which it reaches the thyrotropin-producing cells of the anterior pituitary 35 .Thyroid hormone secretion is suppressed during starvation, while preprohypocretin mRNA is upregulated in the LHA 36 .We hypothesized that hypocretin neural circuits can modulate the HPT axis.Despite the evolving understanding of the hypocretin system, its role on the HPT axis in NT1 seems to remain unclear.

Thyroid dysfunction and depression
The neuroendocrine dysfunction theory of the pathogenesis of depression mainly includes abnormalities of the hypothalamic-pituitary-adrenal axis and the HPA axis 10 .The relationship between thyroid function and depression has been of great interest to scholars, and it has been suggested that there are alterations in the function of the hypothalamic pituitary thyroid (HPT) axis in patients with depression, and it has been reported that www.nature.com/scientificreports/disturbances in the level of thyroid hormones not only affects the patient's emotional response, but also serves as a marker of sensitivity in some patients with depression 15 .Studies have indicated that FT3 and FT4 may be the hallmark markers for predicting the degree of depression, and thyroid-related tests may be considered as routine examinations for adolescents with depression 16 .Hypothyroidism can induce depression and inattention, while mood disorders also be able to modulate the function of HPT axis 16 .Some researchers have proposed that thyroid hormones have an antidepressant effect in depression 37 .The serum TSH level of patients with NT1 was linked with the more severe subjective sleepiness symptoms, and the worse depression.In addition, we found that the serum FT 4 levels was positively correlated with HAMA 14 score.Hypocretin deficiency can induce the cholinergic monoaminergic imbalance, which may be involved in the pathophysiological process of depression 18 .However, although previous studies have found that thyroid hormones are involved in the pathogenesis of depression, the exact mechanism of the HPT axis in depression remains unclear.

Limitations and recommendations
Firstly, subjects should be followed up periodically to assess their clinical characteristics as well as thyroid function, further illustrating the changes in thyroid hormones in patients with NT1.In addition, our results remain weak due to the sample size; our study subjects represent only NT1 patients with cataplexy.

Conclusion
Hypocretin deficiency may be associated with the regulation of thyroid hormones in NT1 patients.The serum thyroid hormones may affect the severity and neuropsychological functions of NT1 patients.The exact relationship between thyroid hormones and NT1 should be validated in multi-center, and large-sample clinical studies in the future.

Figure 1 .
Figure 1.Flow chart of patient inclusion.

Table 1 .
Demographic data and results of biochemical analyses.Data are presented as mean ± standard deviation, or median (interquartile range) as appropriate.The differences were considered significant if p-value < 0.05.BMI body mass index defined as weight in kilograms divided by the square of height in meters, SBP systolic blood pressure, DBP diastolic blood pressure, PSG polysomnography, AHI apnea hypopnea index, PLMI rapid eye movement sleep latency, ESS Epworth sleepiness scale, PSQI Pittsburgh sleep quality index (PSQI) scale, HAMD 24 24-item Hamilton depression rating scale, HAMA 14 , 14-item Hamilton anxiety scale.a χ 2 -test, b t test.